Reactive Oxygen Species Production Is a Major Factor Directing the Postantibiotic Effect of Fluoroquinolones in Streptococcus pneumoniae

Abstract

We studied the molecular mechanisms involved in the postantibiotic effect of the fluoroquinolones levofloxacin and moxifloxacin in Streptococcus pneumoniae. Wild-type strain R6 had postantibiotic effects of 2.05 Ϯ 0.10 h (mean Ϯ standard deviation [SD]) and 3.23 Ϯ 0.45 h at 2.5ϫ and 10ϫ MIC of levofloxacin, respectively. Moxifloxacin exhibited lower effects of 0.87 Ϯ 0.1 and 2.41 Ϯ 0.29 h at 2.5ϫ and 10ϫ MIC, respectively. Fluoroquinolone-induced chromosome fragmentation was measured at equivalent postantibiotic effects for levofloxacin (2.5ϫ MIC) and moxifloxacin (10ϫ MIC). After 2 h of drug removal, reductions were approximately 7-fold for levofloxacin and 3-fold for moxifloxacin, without further decreases at later times. Variations in reactive oxygen species production were detected after 4 to 6 h of drug withdrawals, with decreases Ն400-fold for levofloxacin and Ն800-fold for moxifloxacin at 6 h. In accordance, after 4 to 6 h of drug withdrawal, the levofloxacin-induced upregulation of the fatCDEB operon, introducing iron in the bacteria, decreased up to 2-to 3-fold, and the moxifloxacin-induced upregulation of several genes involved in the production of pyruvate was reduced 3-to 7-fold. In accordance, lower postantibiotic effects (up to 1 h) were observed in strain R6 ΔspxB, lacking the main enzyme involved in oxygen peroxide production, than in R6. Although no change in the recovery of chromosome fragmentation was observed between R6 and R6 ΔspxB, 3.5 ϫ 10 3 -fold lower reactive oxygen species production was observed in R6 ΔspxB, without changes after drug removal. These results show that reactive oxygen species are the main factors directing the postantibiotic effect of levofloxacin and moxifloxacin in S. pneumoniae.

Pneumococcal Evasion of Antibiotics via Metabolic Adaptation During Infection

“…In our study, the population sequencing and functional characterization of the evolved populations indicated an important role of hydroxyl radicals for the observed tolerance phenotypes, a finding subsequently confirmed via targeted mutagenesis and chemical inhibition. This is in strong agreement with previous observations, whereby reactive oxygen species production is a critical aspect of the post-antibiotic effect for delayed regrowth of fluroquinolone treated S. pneumoniae 69 . Additional support for the role of oxidative stress in potentiation of fluroquinolone killing comes from the observation that under levofloxacin stress, the pneumococcus upregulates genes involved in iron transport, whose substrate can subsequently generate oxidative stress through the Fenton reaction 83 .…”

“…This result suggests that, in the absence of fluoroquinolone-induced free radicals, the pneumococcus can become tolerant to fluoroquinolone killing. As both redox stress and fluroquinolones can result in DNA damage and fragmentation that can contribute to bactericidal activity 68, 69 , we next sought to determine whether the protection engendered by edaravone correlated with a corresponding decrease in DNA fragmentation during fluroquinolone treatment. While treatment with either levofloxacin or ciprofloxacin resulted in elevated DNA fragmentation ( Figure 7C and D ), this effect was completely abrogated upon treatment with edaravone, bringing the antibiotic exposed cells to basal wild-type levels ( Figure 7C and D ).…”

The TLR4-MyD88 Signaling Axis Regulates Lung Monocyte Differentiation Pathways in Response to Streptococcus pneumoniae

“…Moreover, NADPH defects in humans favor fungal and bacterial infection ( 61 ). Likewise, the bactericidal activity of the fluoroquinolones levofloxacin and moxifloxacin against S. pneumoniae is related to the production of ROS by the bacteria, associated with transcriptional alterations induced by these fluoroquinolones ( 64 – 66 ). Moreover, oxygen-derived metabolites can also damage infected tissues, regulate immune functions and induce apoptosis ( 58 , 67 ).…”

Antibacterial activity of a DNA topoisomerase I inhibitor versus fluoroquinolones in Streptococcus pneumoniae

“…Some studies, which support our results, have reported that PAE induced by LVX [58] or MXF [59] in planktonic cultures of S. pneumoniae. We have recently studied the molecular mechanisms involved in this process and found that the production of reactive oxygen species, which is a consequence of transcriptional alterations induced by the drugs, is a major PAE contributor [60]. However, PAE has never been studied in biofilms.…”